This invention relates to methods for the preparation of diastereomerically enriched phosphorothioate linked oligonucleotides, and to intermediates useful in their preparation. This invention also relates to sequence-specific phosphorothioate oligonucleotides having chiral phosphorus linkages and to a novel chemical synthesis of these and other oligonucleotides.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect might be obtained with minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides. Oligonucleotides complementary to a specific target messenger RNA (mRNA) sequence are used. Several oligonucleotides are currently undergoing clinical trials for such use.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate the action of transcription factors. Several recent reports describe such interactions (see, Bielinska, et. al., Science 1990, 250, 997-1000; and Wu, et al., Gene 1990, 89, 203-209.)
Oligonucleotides also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with the above gene expression inhibition, diagnostic use can take advantage of an oligonucleotide""s ability to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of such other biological molecules. One particular use, the use of oligonucleotides as primers in the reactions associated with polymerase chain reaction (PCR), has been the cornerstone for the establishment of an ever expanding commercial business. The use of such PCR reactions has seemingly xe2x80x9cexplodedxe2x80x9d as more and more use of this very important biological tool is made. The uses of PCR have extended into many areas in addition to those contemplated by its Nobel laureate inventor. Examples of such new areas include forensics, paleontology, evolutionary studies and genetic counseling to name just a few. Primers are needed for each of these uses. Oligonucleotides, both natural and synthetic, serve as the primers.
Oligonucleotides also are used in other laboratory procedures. A number of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et. al., Eds., Current Publications, 1993. Such uses include Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA from Book 2 of Molecular Cloning, A Laboratory Manual, ibid. and DNA-Protein Interactions and The Polymerase Chain Reaction from Vol. 2 of Current Protocols In Molecular Biology, ibid.
To supply the users of oligonucleotides, many scientific journals now contain advertisements for either oligonucleotide precursors or for custom-synthesized oligonucleotides. This has become an important commercial use of oligonucleotides. Oligonucleotides can be synthesized to have properties that are tailored for the desired use. Thus, a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents, and as therapeutic entities. These modifications are designed, for example, to increase binding to a target nucleic acid strand, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to provide stability against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, or to improve the pharmacokinetic properties of the oligonucleotides.
Since they exist as diastereomers, phosphorothioate, methylphosphonate, phosphotriester, phosphoramidate and other phosphorus oligonucleotides synthesized using known, automated techniques result in mixtures of Rp and Sp diastereomers at the individual phosphorothioate, methylphosphonate, phosphotriester, phosphoramidate or other phosphorus linkages. Thus, a 15-mer oligonucleotide containing 14 asymmetric linkages has 214, i.e. 16,384, possible stereoisomers. It is possible that oligomers having diastereomerically enriched linkages could possess advantages in hybridizing to a target mRNA or DNA. Accordingly, there is a need for such oligomers.
Miller, P. S., McParland, K. B., Jayaraman, K., and Ts""o, P.O.P (1981), Biochemistry, 20:1874, found that small di-, tri- and tetramethylphosphonate and phosphotriester oligonucleotides hybridize to unmodified strands with greater affinity than natural phosphodiester oligonucleotides. Similar increased hybridization was noted for small phosphotriester and phosphoramidate oligo-nucleotides; Koole, L. H., van Genderen, M. H. P., Reiners, R. G., and Buck, H. M. (1987), Proc. K. Ned. Adad. Wet., 90:41; Letsinger, R. L., Bach, S. A., and Eadie, J. S. (1986), Nucleic Acids Res., 14:3487; and Jager, A., Levy, M. J., and Hecht, S. M. (1988), Biochemistry, 27:7237. The effects of the diastereomers of undefined stereochemistry on hybridization becomes even more complex as chain length increases.
Bryant, F. R. and Benkovic, S. J. (1979), Biochemistry, 18:2825 studied the effects of diesterase on the diastereomers of ATP. Published patent application PCT/US88/03634 discloses dimers and trimers of 2xe2x80x2,5xe2x80x2-linked diastereomeric adenosine units. Niewiarowski, W., Lesnikowski, Z. J., Wilk, A., Guga, P., Okruszek, A., Uznanski, B., and Stec, W. (1987), Acta Biochimica Polonia, 34:217, synthesized dimers of thymidine having high diastereomeric excess, as did Fujii, M., Ozaki, K., Sekine, M., and Hata, T. (1987), Tetrahedron, 43:3395.
Stec, W. J., Zon, G., and Uznanski, B. (1985), J. Chromatography, 326:263, have reported the synthesis of certain mixtures of phosphorothioates or methyphosphonate oligonucleotides and have separated them by chromatography. However, they were only able to separate the diastereomers of certain small oligomers having a limited number of diastereomerically pure phosphorus linkages.
In a preliminary report, J. W. Stec, Oligonucleotides as antisense inhibitors of gene expression: Therapeutic implications, meeting abstracts, Jun. 18-21, 1989, noted that a non-sequence-specific thymidine homopolymer octamerxe2x80x94i.e. a (dT)8-mer, having xe2x80x9call-except-onexe2x80x9d Rp configuration methylphosphonate linkagesxe2x80x94formed a thermodynamically more stable hybrid with a 15-mer deoxyadenosine homopolymerxe2x80x94i.e. a d(A)15-merxe2x80x94than did a similar thymidine homopolymer having xe2x80x9call-except-onexe2x80x9d Sp configuration methylphosphonate linkages. The hybrid between the xe2x80x9call-except-onexe2x80x9d Rp (dT)8-mer and the d(A)15-mer had a Tm of 38xc2x0 C. while the Tm of the xe2x80x9call-except-onexe2x80x9d Sp (dT)8-mer and the d(A)15-mer was  less than 0xc2x0 C. The hybrid between a (dT)8-mer having natural phosphodiester linkages, i.e. octathymidylic acid, and the d(A)15-mer was reported to have a Tm of 14xc2x0 C. The xe2x80x9call-except-onexe2x80x9d thymidine homopolymer octamers were formed from two thymidine methylphosphonate tetrameric units with high diastereomeric excess linked by a natural phosphodiester linkage.
Six or more nucleotides units are generally necessary for an oligonucleotide to be of optimal use in applications involving hybridization. It is often preferred to have even more nucleoside units for best performance, often as many as 10 to 30. Because it has not been possible to stereochemically resolve more than two or three adjacent phosphorus linkages, the effects of induced chirality in the phosphorus linkages of chemically synthesized oligonucleotides has not been well assessed heretofore. This is because with few limited exceptions, the sequence-specific phosphorothioate, methylphosphonate, phosphotriester or phosphoramidate oligonucleotides obtained utilizing known automated synthetic techniques have been mixtures with no diastereomeric excess.
Some aspects of the use of enzymatic methods to synthesize oligonucleotides having chiral phosphorus linkages have been investigated. Burgers, P. M. J. and Eckstein, F. (1979), J. Biological Chemistry, 254:6889; and Gupta, A., DeBrosse, C., and Benkovic, S. J. (1982) J. Bio. Chem., 256:7689 enzymatically synthesized diastereomerically pure polydeoxyadenylic acid having phosphorothioate linkages. Brody, R. S. and Frey, P. S. (1981), Biochemistry, 20:1245; Eckstein, F. and Jovin, T. M. (1983), Biochemistry, 2:4546; Brody, R. S., Adler, S., Modrich, P., Stec, W. J., Leznikowski, Z. J., and Frey, P. A. (1982) Biochemistry, 21: 2570-2572; and Romaniuk, P. J. and Eckstein, F. (1982) J. Biol. Chem., 257:7684-7688 all enzymatically synthesized poly TpA and poly ApT phosphorothioates while Burgers, P. M. J. and Eckstein, F. (1978) Proc. Natl. Acad. Sci. USA, 75: 4798-4800 enzymatically synthesized poly UpA phosphorothioates. Cruse, W. B. T., Salisbury, T., Brown, T., Cosstick, R. Eckstein, F., and Kennard, O. (1986), J. Mol. Biol., 192:891, linked three diastereomeric Rp GpC phosphorothioate dimers via natural phosphodiester bonds into a hexamer. Most recently Ueda, T., Tohda, H., Chikazuni, N., Eckstein, R., and Watanabe, K. (1991) Nucleic Acids Research, 19:547, enzymatically synthesized RNA""s having from several hundred to ten thousand nucleotides incorporating Rp linkages of high diastereomeric excess. Enzymatic synthesis, however, is disadvantageous in that it depends on suitable polymerases that may or may not be available, especially for modified nucleoside precursors.
As reviewed by W. J. Stec and A. Wiek (1994), Angew. Chem. Int. Ed. English 33:709, the oxathiaphospholane method has been successful for the preparation of phosphorothioates with defined stereochemistry. However, it suffers from disadvantages, such as the non-trivial preparation of diastereomerically pure oxathiaphospholane, and the difficulty in synthesizing and isolating satisfactorily pure oligomers longer than 12-mers.
It would therefore be of great advantage to provide oligonucleotides having phosphorus linkages with controlled stereochemistry.
It is one object of this invention to provide sequence-specific oligonucleotides having chirally pure phosphorothioate linkages with high diastereomeric excess.
Another object is to provide phosphorus-linked oligonucleotides having substantially all Rp or all Sp linkages.
A further object is to provide research and diagnostic materials for assaying bodily states in animals, especially diseased states.
It is yet another object to provide new methods for synthesizing sequence-specific oligonucleotides having chirally pure phosphorothioate linkages, and useful intermediates therefor.
The present invention provides stereoselective methods for preparing sequence-specific oligonucleotides having chiral phosphorus linkages. In certain preferred embodiments, these methods comprise the steps of:
reacting a first synthon of Formula I: 
xe2x80x83wherein:
Q is independently O or S;
R1 is a hydroxyl protecting group;
R2 is a chiral auxiliary of formula xe2x80x94C(R8)R3xe2x80x94C(R16)R5xe2x80x94CHR6xe2x80x94NHR7;
R3 is hydrogen, alkyl, cyanomethyl, monohalomethyl, dihalomethyl, trihalomethyl, xe2x80x94CH2R4, xe2x80x94CH2Si(R4)3, or xe2x80x94CH2xe2x80x94SOkR4 where k is 0, 1 or 2;
R4 is independently alkyl, aryl, aralkyl or alkaryl having up to 15 carbon atoms, xe2x80x94N(R70)xe2x80x94C(xe2x95x90O)xe2x80x94R71, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94R70, or xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94N(R70)(R71);
R70 and R71 are each independently alkyl, xcex1-halo substituted alkyl, aralkyl, xcex1-halo substituted aralkyl, or aryl substituted with up to three electronegative groups;
R5 is H, xe2x80x94CN, xe2x80x94Si(R4)3, SOkR4 or halogen;
or R8 and R16 are each H, and R3 and R5, together, form one of the structures: 
xe2x80x83wherein:
R10 and R11 are H, alkyl having from 1 to about 10 carbons, xe2x80x94CH2C(xe2x95x90O)OR22, xe2x80x94CH2CN, xe2x80x94CH2Si(CH3)3, or o- or p-C6H4xe2x80x94R21;
R21 is hydrogen, xe2x80x94Oxe2x80x94C(xe2x95x90O)CH3, alkoxy having from 1 to about 10 carbons, xe2x80x94NO2, or xe2x80x94N(R22)2;
R22 is independently H or alkyl having from one to about 10 carbon atoms;
p is 1 or 2;
Z1 and Z2 are independently halogen, xe2x80x94CN, xe2x80x94Si(CH3)3, or xe2x80x94C(xe2x95x90O)OR22;
R30 is hydrogen, xe2x80x94Oxe2x80x94C(xe2x95x90O)CH3, alkoxy having from 1 to about 10 carbons, or xe2x80x94Oxe2x80x94Si(R4)3;
R6 is H, alkyl or aralkyl having up to 15 carbon atoms;
or R5 and R6, together with the atoms to which they are attached, form a 5 or 6 membered ring;
R7 is alkyl or aralkyl having up to 15 carbon atoms;
or R6 and R7, together, form one of the structures 
xe2x80x83wherein
V, T, and Z are independently CH or N;
R41, R42, R43, and R44 are each independently H or an electronegative group;
R8 is H or methyl;
R16 is H, alkyl or aralkyl having up to 15 carbon atoms;
B is a nucleobase; and
n is an integer from 0 to 50;
xe2x80x83with a second synthon of Formula II: 
xe2x80x83wherein:
R9 is a hydroxyl protecting group or a linker connected to a solid support; and
m is an integer from 0 to 50;
xe2x80x83for a time and under reaction conditions effective to form a third synthon of Formula III: 
xe2x80x83and
contacting said third synthon with a sulfurizing agent to form an oligomer of Formula IV: 
wherein D is said phosphorothioate linkage having the formula: 
In preferred embodiments, said phosphorothioate linkage is diastereomerically enriched. In other preferred embodiments about 75% of the phosphorothioate linkage is in a single stereoisomeric form. In further preferred embodiments about 85% of the phosphorothioate linkage is in a single stereoisomeric form. In especially preferred embodiments about 95% of the phosphorothioate linkage is in a single stereoisomeric form. Most preferably, the phosphorothioate linkage is in a single stereoisomeric form, substantially free of other stereoisomeric forms. Preferably, the first synthon is in a single stereoisomeric form, substantially free of other stereoisomeric forms.
In some preferred embodiments n is 0. In further preferred embodiments, R1 groups are subsequently removed to yield new second synthons for iterative synthesis, and chiral auxiliaries are removed after iterative synthesis is completed. In preferred embodiments of the present methods the oligomer of Formula IV contains a plurality of phosphorothioate linkages.
Preferably, first and second synthons are reacted at a temperature of from about xe2x88x9220xc2x0 C. to about 40xc2x0 C, with from about xe2x88x9215xc2x0 C. to about 0xc2x0 C. being more preferred.
In some preferred embodiments the first synthon is formed by reacting a compound of Formula V: 
with an azaphospholane of Formula VIa: 
wherein R3xe2x80x94R8 are as defined above; and X is halogen, dialkylamino, imidazole, triazole or substituted phenoxy wherein said substituents are electron withdrawing, preferably halogen or nitro.
In some embodiments the azaphospholane described above is produced by reacting a reagent of formula HOxe2x80x94C(R8)R3xe2x80x94C(R16)R5xe2x80x94CHR6xe2x80x94NHR7 and a phosphorus trihalide, phosphorus tri(dialkylamide), phosphorus triphenoxide or phosphorus triimidazolide.
In more preferred embodiments the first synthon is formed by reacting a compound of Formula VII: 
and a xcex3-amino alcohol of formula HOxe2x80x94C(R8)R3xe2x80x94C(R16)R5xe2x80x94CHR6xe2x80x94NHR7. Preferably, X is chlorine, dialkylamino or diphenoxy, and said reaction is stereoselective. It is especially preferred that the first synthon is in a single stereoisomeric form, substantially free of other stereoisomeric forms.
In some preferred embodiments the reaction of first and second synthons is performed in the presence of a catalyst, said catalyst preferably having one of the Formulas VIII or IX: 
wherein:
R12 and R13 are independently hydrogen, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, an ester group, or R12 and R13 together with the carbon atoms to which they are attached, form a substituted or unsubstituted phenyl ring where said substituents are electron withdrawing; and
R14 is hydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, norbornyl, substituted norbornyl, aryl, substituted aryl wherein said substituents are electron withdrawing, or has the formula: 
wherein L is protecting group.
In some preferred embodiments R14 is halogen or nitro, preferably bromine, and R12 and R13 are each halogen or each cyano, with cyano being especially preferred.
Other preferred embodiments R14 has one of the formulas: 
wherein R15 is H, methyl, trialkylsilyl or acetyl.
In some preferred embodiments of the method R3 is cyanomethyl or xe2x80x94CH2xe2x80x94SOkR4 where k is 0, 1 or 2, and R7 is lower alkyl or aralkyl.
In further preferred embodiments said first synthon has one of the Formulas Xa, XIa, XIIa, XIIIa or XXa: 
wherein W has the formula: 
and R1xe2x80x94R 16, V, T and Z are as defined above.
Other preferred first synthons have the Formula Xb or Xc: 
More preferred first synthons have the Formula XVIIa or XVIIIa: 
Particularly preferred first synthons have the Formula XIVa: 
Especially preferred first synthons have the Formula XVa or XVIa: 
In preferred embodiments of the methods of the invention R1 groups are removed from the oligomers, thus creating new second synthons for further iterative synthesis.
Also provided according to the invention are phosphorothioate oligomers produced by the method of claim 1, and azaphospholanes having Formula VIb: 
In preferred embodiments of the invention, 75% of said azaphospholanes having Formula VIb are in a single stereoisomeric form, with 85% being more preferred, and 95% being particualrly preferred. In especially preferred embodiments, the azaphospholanes having Formula VIb are in a single stereoisomeric form, substantially free of other stereoisomeric forms.
In preferred embodiments the azaphospholane has one of the Formulas Xb, XIb, XIIb, XIIIb, Xd, Xe or XXb: 
In other preferred embodiments the azaphospholane has the Formula XVIIb or XVIIIb: 
In some particularly preferred embodiments the azaphosphalane has the Formula XIVb: 
Especially preferred embodiments the azaphospholane has the Formula XVb or XVIb: 
Also provided in accordance with the invention are oligomeric compounds comprising a phosphite linkage having the Formula XXX: 
In preferred embodiments of the invention, 75% of said phosphosphite linkage is in a single stereoisomeric form, with 85% being more preferred, and 95% being particualrly preferred. In especially preferred embodiments, the phosphosphite linkage is in a single stereoisomeric form, substantially free of other stereoisomeric forms.
The present invention is directed to methods for the synthesis of phosphorothioate compounds having diastereomerically enriched phosphorothioate linkages, and to intermediates useful in their preparation.
In one aspect, the invention provides methods for the preparation of phosphorothioate linkages comprising the steps of reacting a first synthon of Formula I: 
wherein:
Q is independently O or S;
R1 is a hydroxyl protecting group;
R2 is a chiral auxiliary of formula xe2x80x94C(R8)R3xe2x80x94C(R16)R5xe2x80x94CHR6xe2x80x94NHR7;
R3 is hydrogen, alkyl, cyanomethyl, monohalomethyl, dihalomethyl, trihalomethyl, xe2x80x94CH2R4, xe2x80x94CH2Si(R4)3, or xe2x80x94CH2xe2x80x94SOkR4 where k is 0, 1 or 2;
R4 is independently alkyl, aryl, aralkyl or alkaryl having up to 15 carbon atoms, xe2x80x94N(R70)xe2x80x94C(xe2x95x90O)xe2x80x94R71, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94R70, or xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94N(R70)(R71);
R70 and R71 are each independently alkyl, xcex1-halo substituted alkyl, aralkyl, xcex1-halo substituted aralkyl, or aryl substituted with up to three electronegative groups;
R5 is H, xe2x80x94CN, xe2x80x94Si(R4)3, SOkR4 or halogen;
or R8 and R16 are each H, and R3 and R5, together, form one of the structures 
xe2x80x83wherein:
R10 and R11 are H, alkyl having from 1 to about 10 carbons, xe2x80x94CH2C(xe2x95x90O)OR22, xe2x80x94CH2CN, xe2x80x94CH2Si(CH3)3, or o- or p-C6H4xe2x80x94R21;
R21 is hydrogen, xe2x80x94Oxe2x80x94C(xe2x95x90O)CH3, alkoxy having from 1 to about 10 carbons, xe2x80x94NO2, or xe2x80x94N(R22)2;
R22 is independently H or alkyl having from one to about 10 carbon atoms;
p is 1 or 2;
Z1 and Z2 are independently halogen, CN, xe2x80x94Si(CH3)3, and xe2x80x94C(xe2x95x90O)OR22;
R30 is hydrogen, xe2x80x94Oxe2x80x94C(xe2x95x90O)CH3, alkoxy having from 1 to about 10 carbons, or xe2x80x94Oxe2x80x94Si(R4)3;
R6 is H, alkyl or aralkyl having up to 15 carbon atoms;
or R5 and R6, together with the atoms to which they are attached, form a 5 or 6 membered ring;
R7 is alkyl or aralkyl having up to 15 carbon atoms;
or R6 and R7, together, form one of the structures 
xe2x80x83wherein
V, T, and Z are independently CH or N;
R41, R42, R43, and R44 are each independently H or an electronegative group;
R8 is H or methyl;
R16 is H, alkyl or aralkyl having up to 15 carbon atoms;
B is a nucleobase; and
n is an integer from 0 to 50;
with a second synthon of Formula II: 
wherein:
R9 is a hydroxyl protecting group or a linker connected to a solid support; and
m is an integer from 0 to 50;
for a time and under reaction conditions effective to form a third synthon of Formula III: 
and
contacting said third synthon with a sulfurizing agent to form an oligomer of Formula IV: 
wherein D is said phosphorothioate linkage having the formula: 
In accordance with the invention, first synthons are cyclic phosphoramidites having the general Formula VIc: 
in which W, R3, R5xe2x80x94R8 and R16 are as defined above.
The reaction of first and second synthons is conducted in the presence of a catalyst. The structures of the first synthon and the catalyst are chosen such that the opening of the cyclic Oxe2x80x94Pxe2x80x94N phosphoramidite (azaphospholane) ring proceeds by the stereoselective breaking of the intracyclic Pxe2x80x94N bond of the azaphospholane, to yield a third synthon, which is diastereomerically enriched at phosphorus. Accordingly, in preferred embodiments of the methods of the invention, first synthons are diastereomerically enriched, and more preferably in a single stereochemical form, substantially free of other stereochemical forms. It is also advantageous for the first synthon and the catalyst to bear substituent groups which are of relatively large size (i.e., bulky groups) to aid in the proper orientation of reactants to achieve the desired stereoselectivity. As used herein, the term stereoselective has its normal meaning as a process in which one stereoisomer is produced or destroyed more rapidly than another, resuting in a predominance of the favored stereoisomer.
In preferred embodiments catalysts have one of the Formulas VIII or IX: 
wherein R12xe2x80x94R14 are as defined above.
It has been found in accordance with the present invention that imidazole catalysts having electron-withdrawing substituents, in addition to substituents of relatively large size, are especially advantageous in production of stereochemically enriched products. While not wishing to be bound by a particular theory, it is believed that the catalyst first protonates the azaphospholane nitrogen, creating a good leaving group, which is displaced by the catalyst or its conjugate base. The imidazole or tetrazole attached to the phosphorus is then displaced either by the 3xe2x80x2-hydroxyl of the nucleosidic species, leading to a phosphite triester of high stereochemical purity, or by the catalyst, leading to epimerization.
It has been found in accordance with the present invention that catalysts which have appreciable acidity (i.e., which have pKa values of about 2 to 4) and which are relatively large can overcome the tendency toward epimerization at phosphorus, and result in stereoselective addition of the free 5xe2x80x2-hydroxyl of the nucleosidic species to be added. Thus, preferred substituents for groups R13, R14 and R15 are those which are electron withdrawing, (and which therefore increase acidity), and of a size sufficient to maintain stereoselectivity. It will be recognized, however, that it is not necessary that all three groups R6, R7 and R8 be of great bulk, so long as the overall size of the catalyst is sufficient to afford the desired stereoselectivity. Thus preferred R12 and R13 groups are independently hydrogen, halogen, cyano, nitro,. alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, an ester group, or R12 and R13, together with the carbon atoms to which they are attached, form a substituted or unsubstituted phenyl ring where said substituents are electron withdrawing. Preferred R14 groups include hydrogen, halogen, cyano, nitro, thio, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, norbornyl, substituted norbornyl, aryl, substituted aryl wherein said substituents are electron withdrawing, or has the formula: 
wherein L is protecting group. In prefered embodiments of the invention the catalyst is 2,4,5-tribromoimidazole, dibromocyanoimidazole, or dicyanobromoimidazole. In particularly preferred embodiments the catalyst is 4,5-dicyano-2-bromoimidazole.
It has been found in accordance with the present invetion that the dicyanoimidazole, bromoimidazole, and tribromoimidazole catalysts described in accordance with the present invention are useful as substitutes for tetrazole catalysts in standard solid phase oligonucleotide synthetic regimes. Such synthetic procedures are well known in the art, and are extensively described in the literature. See for example, Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. No. 4,725,677 and U.S. Pat. No. Re. 34,069, and Oligonucleotides and Analogues, A Practical Approach, Eckstein, F., IRL Press, New York (1991). The use of the catalysts of the invention in these synthetic methologies provides significant advantages over tetrazole catalysts, including, for example, significantly lower cost.
In other preferred embodiments R14 has one of the formulas: 
wherein R15 is H, methyl, trimethylsilyl or acetyl.
In some preferred embodiments R6 and R7, together with the atoms to which they are attached, form an heterocyclic (i.e., imidazole, triazole or tetrazole) ring, which performs the function of the catalyst. Preferred first synthons which incorporate the catalyst therein have the general Formula Xa or XIIIa: 
wherein V, T and Z are each independently N or CH. In especially preferred embodiments the first synthons incorporate imidazole rings, and have the Formula Xb or Xc: 
In further preferred embodiments of the invention the imidazole portions of the first synthons are further substituted, for example, by having a phenyl ring fused thereto, which preferably bears one or more electronegative groups. Thus in another preferred embodiment first synthons have the Formula XIa: 
In further preferred embodiments, first synthons incorporate other relatively large substituent groups which facilitate the stereoselective opening of the azaphospholane ring. In particularly preferred embodiments first synthons have the Formula XIIa, and particularly Formula XVIIa or XVIIIa: 
in which R10 and R11 are as defined above.
In especially preferred embodiments, first synthons have the Formula XVb or XVIb: 
In some preferred embodiments, the first synthon is obtained by reaction of a compound of Formula V: 
with an azaphospholane of Formula VIa: 
wherein R3xe2x80x94R8 are as defined above; and X is halogen, preferably chlorine, dialkylamino, imidazole or substituted phenoxy wherein said substituents are electron withdrawing, and preferably are halogen or nitro.
In more preferred embodiments the first synthon is obtained by reaction of a compound of Formula VII: 
and a xcex3-amino alcohol of formula HOxe2x80x94C(R8)R3xe2x80x94C(R16)R5xe2x80x94CHR6xe2x80x94NHR7; wherein X and R1xe2x80x94R16 are as defined above.
R2 is a chiral auxiliary, which has the formula xe2x80x94C(R8)R3xe2x80x94C(R16)R5xe2x80x94CHR6xe2x80x94NHR7, and which is formed as a consequence of the opening of the cyclic phosphite ring. The chiral auxiliary functions as a protecting group for the phosphorus linkage during the course of the synthesis of oligomeric phosphorothioates. Accordingly, chiral auxiliaries are allowed to remain on the growing chain, and are removed at the end of the iterative synthetic regime. Removal of chiral auxiliaries can be conveniently accomplished in a single treatment after the completion of the iterative synthesis by treatment with either acidic reagents or by base catalyzed xcex2-elimination. Suitable acidic reagents include xcex1-halo organic acids such as, for example, 70% trifluoroacetic acid. Suitable reagents for removing chiral auxiliaries by xcex2-elimination include ammonia and fluoride ion. Removal of chiral auxiliaries via xcex2-elimination should be particularly advantageous where first synthons have the Formula XXa or Formula XIa, especially where at least one of R41, R42, R43, and R44 is an electronegative group, or where R3 is xe2x80x94CH2R4, R4 is independently xe2x80x94N(R70)xe2x80x94C(xe2x95x90O)xe2x80x94R71, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94R70, or xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94N(R70)(R71).
After reacting first and second synthons to form a third synthon, the third synthon is sulfurized to form a phosphorothioate linkage having the formula: 
Sulfurization may be accomplished by any of the several sulfurizing agents known in the art to be suitable for conversion of phosphites into phosphorothioates. Useful sulfurizing agents include Beaucage reagent described in e.g., Iyer, R. P.; Egan, W.; Regan, J. B.; Beaucage, S. L., 3H-1,2-Benzodithiole-3-one 1,1-Dioxide as an Improved Sulfurizing Reagent in the Solid-Phase Synthesis of Oligodeoxyribonucleoside Phosphorothioates, Journal of American Chemical Society, 1990, 112, 1253-1254 and Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan J. B.; Beaucage, S. L., The Automated Synthesis of Sulfur-Containing Oligodeoxyribonucleotides Using 3H-,2-Benzodithiol-3-one 1,1-Dioxide as a Sulfur-Transfer Reagent, Journal of Organic Chemistry, 1990, 55, 4693-4699. Tetraethyl-thiuram disulfide can also be used as described in Vu, H.; Hirschbein, B. L., Internucleotide Phosphite Sulfurization With Tetraethylthiuram Disulfide, Phosphorothioate Oligonucleotide Synthesis Via Phosphoramidite Chemistry, Tetrahedron Letters, 1991, 32, 3005-3007. Further useful reagents for this step are dibenzoyl Tetrasulfide, Rao, M. V.; Reese, C. B.; Zhengyun, Z., Dibenzoyl Tetrasulphidexe2x80x94A Rapid Sulphur Transfer Agent in the Synthesis of Phosphorthioate Analogues of Oligonucleotides, Tetrahedron Letters, 1992, 33, 4839-4842; di(phenylacetyl)disulfide, Kamer, R. C. R.; Roelen, H. C. P. F.; van den Eist, H.; van der Marel, G. A.; van Boom, J. H., An Efficient Approach Toward the Synthesis of Phosphorothioate Diesters Va the Schonberg Reaction, Tetrahedron Letters, 1989, 30, 6757-6760; sulfur; and sulfur in combination with ligands like triaryl, trialkyl or triaralkyl or trialkaryl phosphines.
The methods of the present invention can also be used to produce analogs of phosphorothioates, including phosphoroselenoates and phosphoroboronates. For example, phosphoroselenoates can be prepared by the methods of the invention by utilizing potassium selenocyanate in place of the sulfurizing agents described above. Phosphoroboronates can be prepared by similar adaptation of oxidizing agents known known in the art. See, for example, Antisense Research and Applications, Crooke, S. T., and Lebleu, B., Eds. CRC Press, Boca Raton, Fla. (1993).
R9 and R1 can each be a hydroxyl protecting group. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. The tert-butyldimethylsilyl (TBDMS) group is representative of protecting groups useful for protecting the hydroxyl functionality. A preferred protecting group for R1 is the dimethoxytrityl group. Other representative groups may be found in Greene, T. W. and Wuts, P. G. M., xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d 2d. Ed., Wiley and Sons, 1991. Typically, protecting groups are removed at the end of the iterative synthesis.
R9 may alternatively be a linker connected to a solid support. Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. No. 4,725,677 and Re. 34,069. Linkers are known in the art as short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in Oligonucleotides And Analogues A Pratical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.
Alkyl groups according to the invention include straight chain, branched, and cyclic carbon and hydrogen containing groups such as methyl, isopropyl, and cyclohexyl groups. Preferred alkyl groups have 1 to about 6 carbon atoms.
Aralkyl groups according to the invention include both alkyl and aryl portions, although the point of attachment of such groups is through an alkyl portion thereof. Benzyl groups provide one example of an aralkyl group. Alkaryl groups include both alkyl and aryl portions, and are attached through their aryl portions. The term aryl is intended to denote monocyclic and polycyclic aromatic groups including, for example, phenyl, naphthyl, xylyl, pyrrole, and furyl groups. Although aryl groups (e.g., imidazo groups) can include as few as 3 carbon atoms, preferred aryl groups have 6 to about 14 carbon atoms, more preferably 6 to about 10 carbon atoms. The alkyl, alkaryl, and aryl groups may be substituted (e.g., i.e, bear halogens and hydroxy groups) or unsubstituted moieties. In some prefered embodiments the alkyl or aralkyl groups bear electron withdrawing substituents, preferably halogen atoms, in one their xcex1-carbons. Examples of xcex1-halo substituted alkyl groups include mono-, di-, and trihalo methyl groups. Examples of xcex1-halo substituted aralkyl groups include xcex1-halo benzyl groups.
Certain substituent groups of compounds of the invention bear electron withdrawing groups. As used herein, the term xe2x80x9celectron wihdrawingxe2x80x9d has its normal meaning as a chemical functionality which electronically or inductively causes the withdrawal of electron density form the moiety to which the electron withdrawing groups is attached. Representative electron withdrawing groups include nitro groups, halogens, cyano, carboxyl groups and substituted carboxy groups such as ester groups and amido groups. Other electron withdrawing groups will be apparent to those of skill in the art, once armed with the present disclosure.
Substituent B is a nucleobase. The term nucleobase as used herein is intended to include naturally occurring nucleobases (i.e., heterocyclic bases found in naturally occurring nucleic acids) and their non-naturally occurring analogs. Thus, nucleobases according to the invention include naturally occurring bases adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), both in their unprotected state and bearing protecting or masking groups. Examples of nucleobase analogs include N4,N4-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, inosine, N6-isopentyladenine, 1-methyladenine, 2-methylguanine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-thiouracil, 5-(1-propynyl)-2-thiocytosine, 2-thiocytosine, and 2,6-diaminopurine. Other suitable base analogs, for example the pyrimidine analogs 6-azacytosine, 6-azathymidine and 5-trifluoromethyluracil, may be found in Cook, D. P., et al., International Publication No. 92/02258, which is herein incorporated by reference.
The compounds of the invention are preferably up to 50 nucleobases in length, with 10 to 30 nucleobases being more preferred, and 15 to 25 nucleobases being especially preferred.
In preferred embodiments the phosphorothioate linkage produced by the method of the invention is diastereomerically enriched. The term xe2x80x9cdiastereomerically enrichedxe2x80x9d denotes the predominance of one stereochemical form over the other. In preferred embodiments the phosphorothioate linkage is 75% in a single stereochemical form. In further preferred embodiments the phosphorothioate linkage is 85% in a single stereochemical form, with 90% being further preferred and 95% being especially preferred. In further preferred embodiments the phosphorothioate linkage is in a single stereochemical form, substantially free of other stereochemical forms.
Preferably, following sulfurization, the phosphorothioate is next converted to a new first synthon. This is first accomplished by the removal of the 5xe2x80x2-hydroxyl protecting group R1, under conditions which will necessarily depend upon the chemical identity of the specific R1 group. After removal of the protecting group, the unprotected 5xe2x80x2-alcohol may be employed as a new second synthon in the iterative method. Libraries of dimeric and higher synthons may be prepared and stored to facilitate the iterative synthesis of desired nucleobase sequences.
Also provided according to the invention are azaphospholanes of Formula VIb: 
wherein Y is X or W, wherein X is halogen, dialkylamino, imidazole, or substituted phenoxy wherein said substituents are electron withdrawing, and W has the formula: 
wherein constituent members are as defined above. Preferably, the azaphospholanes of Formula VIb are diastereomerically enriched. In particular, it is advantageous to have defined stereochemistry around phosphorus atom, to afford diastereomerically enriched products upon stereoselective opening of the azaphospholane ring.
In preferred embodiments, compounds of the invention have one of the Formulas Xb, XIb, XIIb, XIIIb or XXb: 
wherein R3xe2x80x94R16, Y, V, T, Z, Z1, Z2 and p are as defined above.
Paticularly preferred embodiments of the compounds of the invention have the Formula XIVb, Xd, Xe, XVIIb, XVIIIb, XVb or XVIb: 
As used herein, the term xe2x80x9ccontactingxe2x80x9d means directly or indirectly causing placement together of moieties to be contacted, such that the moieties come into physical contact with each other. Contacting thus includes physical acts such as placing the moieties together in a container. The term xe2x80x9creactingxe2x80x9d as used herein means directly or indirectly causing placement together of moieties to be reacted, such that the moieties chemically combine or transform.
The method of the invention is performed in the presence of a solvent, for example chloroform or acetonitrile. Other solvents suitable for use in the present method will be readily apparent to those skilled in the art, once having been made aware of the present disclosure.
In general, it is preferred that the molar ratio of the catalyst to the first synthon starting material be from about 1 to about 50; preferably from about 2.5 to about 10.
The method of the present invention can be carried out in any suitable vessel which provides efficient contacting between the first and second synthons, and the catalyst. The reaction vessel used should be resistant to the components of the reaction mixture. Glass-lined vessels would be suitable for this purpose. Additional vessel materials will be apparent to those skilled in the art.
The reagents of the present method may be added in any order. The method is preferably carried out under an inert atmosphere, any should be carried out in a dry atmosphere. Any suitable inert gas may be employed, such as nitrogen, helium and argon.
Preferably, the method is carried out at temperatures ranging between about xe2x88x9220xc2x0 C. and about 40xc2x0 C., with temperatures ranging from about xe2x88x9215xc2x0 C. to about 0xc2x0 C. being more preferred.
Reaction time is generally from about one minute to about two hours, with reaction times of from about one minute to about 10 minutes being preferred.
Product can be recovered by any of several methods known to those of skill in the art. Preferably, products are recovered by chromatography. Additional separation of isomers can be accomplished by techniques known in the art including high performance liquid chromatography.
When R9 is a solid support, purification is carried out after removal of the oligonucleotide from the solid support using methods known in the art.